殷杰 赵胜利 蔡晓明 袁辉宗 李俊杰 竺枫

[摘要] 目的 分析基于無翅型MMTV整合位点家族（Wnt）/β-连环蛋白（β-catenin）信号通路，血管内皮生长因子（VEGF）基因转染骨髓源内皮祖细胞（EPCs）移植对大鼠缺血皮瓣的治疗作用。 方法 培养诱导分化大鼠EPCs，脂质体介导VEGF转染EPCs。Wistar大鼠制备缺血皮瓣模型分为对照组、VEGF-EPCs组、NC-EPCs组、Wnt/β-catenin信号通路激活剂氯化锂（LiCl）组。VEGF-EPCs组与NC-EPCs组皮瓣分别注射PcDNA3.1（+）/VEGF165质粒、PcDNA3.1（+）/NC空载对照质粒，对照组注射PBS溶液，LiCl组注射LiCl。酶联免疫吸附实验（ELISA）检测血清VEGF水平;观察大鼠皮瓣存活情况;激光多普勒血液检测仪检测皮瓣蒂部血流;HE染色计算皮瓣毛细血管密度;Western blotting检测皮瓣组织Wnt/β-catenin信号通路相关蛋白表达。 结果 镜下观察分离培养的EPCs外观呈“铺路石”状，经鉴定符合EPCs表面抗原。VEGF-EPCs组、NC-EPCs组、LiCl组血清VEGF水平、皮瓣存活率、PU值、毛细血管密度高于对照组，且VEGF-EPCs组高于NC-EPCs组、LiCl组，差异有统计学意义（P<0.05），NC-EPCs组与LiCl组比较，差异无统计学意义（P>0.05）。VEGF-EPCs组、NC-EPCs组、LiCl组β-catenin、c-myc蛋白相对表达量高于对照组，LiCl组高于VEGF-EPCs组与NC-EPCs组，VEGF-EPCs组高于NC-EPCs组，差异有统计学意义（P<0.05）。 结论 VEGF基因转染骨髓源EPCs移植能促进大鼠缺血皮瓣的存活，改善血液循环，增加微血管密度，其机制与Wnt/β-catenin信号通路的激活有关。

[关键词] 血管内皮生长因子;皮瓣移植;无翅型MMTV整合位点家族;β-连环蛋白;内皮祖细胞

[中图分类号] R622          [文献标识码] A          [文章编号] 1673-9701（2021）31-0036-05

[Abstract] Objective To analyze the therapeutic effects of vascular endothelial growth factor （VEGF） gene transfection of bone marrow-derived endothelial progenitor cells（EPCs） transplantation on ischemic flaps in rats based on the wingless-type mouse mammary tumor virus （MMTV） integration site family （Wnt）/β-catenin signaling pathway. Methods The rat EPCs were cultured and induced to differentiate， and the EPCs were transfected with liposome-mediated VEGF. The Wistar rats were divided into control， VEGF-EPCs， negative control （NC）-EPCs and Wnt/β-catenin signaling pathway activator lithium chloride（LiCl） groups to prepare the ischemic flap models. These Wistar rats in the VEGF-EPCs group， NC-EPCs group， control group and LiCl group were respectively injected with PcDNA3.1（+）/VEGF165 plasmid， PcDNA3.1（+）/NC empty control plasmid， phosphate buffer saline （PBS） solution and LiCl into the skin flaps. Serum VEGF levels were measured by enzyme-linked immunosorbent assay （ELISA）， the survival of rat flaps was observed， blood flow at the tip of the flap was measured by laser Doppler blood detector， HE staining was performed to calculate the capillary density of flap， and Western blotting was adopted to detect the expression of proteins related to Wnt/β-catenin signaling pathway in flap tissue. Results The appearance of EPCs was “paving stone” under microscope， which was identified as meeting the surface antigen of EPCs. Serum VEGF levels， flap survival rate， PU value， and capillary density in the VEGF-EPCs group， NC-EPCs group， and LiCl group were higher than those of the control group， and those of the VEGF-EPCs group were higher than those of the NC-EPCs group and LiCl group，the difference was statistically significant（P<0.05）. However，there were no significant differences between the NC-EPCs group and the LiCl group in the above-mentioned indices （P>0.05）. The relative expression of β-catenin and c-myc protein in the VEGF-EPCs group， NC-EPCs group and LiCl group was higher than that in the control group. Meanwhile，the relative expression of β-catenin and c-myc protein in the LiCl group was higher than that in the VEGF-EPCs group and NC-EPCs group， and that in the VEGF-EPCs group was higher than that in the NC-EPCs group，the difference was statistically significant（P<0.05）. Conclusion VEGF gene transfection of bone marrow-derived EPCs transplantation can promote the survival of ischemic flaps， improve blood circulation and increase microvascular density in rats， and the mechanism is related to the activation of Wnt/β-catenin signaling pathway.

[Key words] Vascular endothelial growth factor; Flap transplantation; Wingless-type MMTV integration site family; β-catenin; Endothelial progenitor cells

皮瓣移植是治疗软组织缺损的有效方式，在肌腱、大血管、神经干、关节、骨等组织裸露的新鲜创面/陈旧性创伤和器官再造中均有应用[1]。移植部位皮瓣血管的新生与血运重建是影响皮瓣存活的关键，干细胞治疗技术的发展，为血运重建的治疗研究提供了新的方向[2]。……